WO2022253353A1 - Pharmaceutical composition comprising hydroxynione and dextromethorphan and application thereof in treating pulmonary fibrosis - Google Patents

Abstract

Disclosed in the present invention are a pharmaceutical composition or pharmaceutical kit comprising hydroxynione or a pharmaceutically acceptable salt thereof and dextromethorphan or a pharmaceutically acceptable salt thereof, and an application thereof in treating pulmonary fibrosis. The hydroxynione or the pharmaceutically acceptable salt thereof and the dextromethorphan or the pharmaceutically acceptable salt thereof, when used in combination, have excellent effects in lung function repair, pulmonary fibrosis inflammation inhibition and pulmonary fibrosis symptom improvement, and therefore, can be used as drugs for treating or alleviating pulmonary fibrosis or improving pulmonary fibrosis symptoms.

Description

Pharmaceutical composition comprising oxynidone and dextromethorphan and its use in treating pulmonary fibrosis

This application claims to enjoy the patent application number 202110627506.5 submitted to the State Intellectual Property Office of China on June 4, 2021, and the title of the invention is "a pharmaceutical composition containing oxynidone and dextromethorphan and its application in the treatment of pulmonary fibrosis" Priority interest in an earlier application. The entirety of said prior application is incorporated by reference into this application.

technical field

The invention belongs to the field of medicaments, and in particular relates to a pharmaceutical composition comprising oxynidone and dextromethorphan and its combination for the preparation of medicine for treating pulmonary fibrosis.

Background technique

Pulmonary fibrosis is the end-stage change of a large class of lung diseases characterized by fibroblast proliferation and accumulation of a large amount of extracellular matrix, accompanied by inflammatory damage and tissue structure destruction, that is, normal alveolar tissue is damaged and undergoes abnormal repair to lead to structural changes. Abnormal (scarring). The cause of most patients with pulmonary fibrosis is unknown (idiopathic). This group of diseases is called idiopathic interstitial pneumonia (IIP), which is a large category of interstitial lung diseases. Among idiopathic interstitial pneumonia (IIP), the most common disease type with pulmonary fibrosis as the main manifestation is idiopathic pulmonary fibrosis (IPF), which is a serious disease that can lead to progressive loss of lung function. interstitial lung disease. Pulmonary fibrosis seriously affects the respiratory function of the human body, manifested as dry cough and progressive dyspnea (perceived insufficient gas), and with the aggravation of the disease and lung damage, the patient's respiratory function continues to deteriorate. The morbidity and mortality of idiopathic pulmonary fibrosis are increasing year by year, and the average survival period after diagnosis is short, and the mortality rate is higher than most tumors, so it is called a "tumor-like disease". Therefore, the development of novel therapeutic drugs for pulmonary fibrosis is expected.

Contents of the invention

In order to improve the above technical problems, the present invention provides a pharmaceutical composition comprising oxynidone represented by formula (I), or a pharmaceutically acceptable salt thereof, and dextromethorphan represented by formula (II) or its pharmaceutically acceptable salts,

The present invention also provides a pharmaceutical kit comprising oxynidone or a pharmaceutically acceptable salt thereof, and dextromethorphan or a pharmaceutically acceptable salt thereof separately.

According to an embodiment of the present invention, in the pharmaceutical composition or pharmaceutical kit, the mass ratio of oxynidone or a pharmaceutically acceptable salt thereof to dextromethorphan or a pharmaceutically acceptable salt thereof may be (1- 10000):(1-5000), such as (10-8000):(1-3000), (10-6000):(1-2000), (10-5000):(1-1000), (50- 4000):(10-500),(100-2000):(50-300),(100-1000):(10-100),(200-1000):(100-200),(300-800) :(100-200),(300-500):(120-180),(1000-5000):(1-800),(2000-5000):(1-600),(2000-5000):( 1-500), (2000-5000): (1-100), (2000-5000): (1-50), (3000-5000): (1-30), (10-5000): (100- 5000), (20-4000):(500-4000), (30-3000):(1000-3000), (50-2000):(1000-3000), (60-1000):(1000-3000) Or (80-800): (1000-3000), based on the active ingredients oxynidone and dextromethorphan.

Those skilled in the art can understand that the mass ratio of oxynidone or a pharmaceutically acceptable salt thereof to dextromethorphan or a pharmaceutically acceptable salt thereof can be any sub-range or specific point value within the aforementioned range.

According to an embodiment of the present invention, the pharmaceutical composition or pharmaceutical kit may further comprise at least one pharmaceutically acceptable excipient. For example, the pharmaceutically acceptable excipients are various pharmaceutical excipients known in the field of pharmacy, including but not limited to: diluents, binders, antioxidants, pH regulators, preservatives, lubricants, disintegrants Wait.

For example, the diluent is selected from lactose, starch, cellulose derivatives, inorganic calcium salts, sorbitol and the like.

For example, the binder is selected from starch, gelatin, sodium carboxymethylcellulose, polyvinylpyrrolidone and the like.

For example, the antioxidant is selected from vitamin E, sodium bisulfite, sodium sulfite, butylated hydroxyanisole and the like.

For example, the pH regulator is selected from hydrochloric acid, sodium hydroxide, citric acid, tartaric acid, Tris, acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate and the like.

For example, the preservative is selected from methylparaben, ethylparaben, m-cresol, benzalkonium chloride and the like.

For example, the lubricant is selected from magnesium stearate, micronized silica gel, talc and the like.

For example, the disintegrant is selected from starch, methylcellulose, xanthan gum, croscarmellose sodium and the like.

According to an embodiment of the present invention, the dosage form of the pharmaceutical composition or pharmaceutical kit can be oral preparations, such as tablets, capsules, pills, powders, granules, suspensions, syrups, etc.; it can also be parenteral administration (For example, injection administration) dosage form, for example, can be injection, powder injection, etc., and it can be through intravenous, intraperitoneal, subcutaneous or intramuscular route.

For example, in the pharmaceutical composition or pharmaceutical kit, the administration methods of the dextromethorphan or a pharmaceutically acceptable salt thereof and oxynidone or a pharmaceutically acceptable salt thereof may be the same or different; for example, both It can be both a parenteral (for example, injection) or oral dosage form, or one of the two is a parenteral (for example, injection) and the other is an oral dosage form; for example In other words, dextromethorphan can be administered parenterally (eg, by injection) and oxynidone as an oral formulation, or vice versa.

The present invention also provides the application of the pharmaceutical composition or pharmaceutical kit in the preparation of medicines for treating or alleviating pulmonary fibrosis or improving symptoms of pulmonary fibrosis.

According to the embodiment of the present invention, the pharmaceutical composition or pharmaceutical kit can restore lung function, inhibit inflammation of pulmonary fibrosis and/or improve fibrosis.

According to the embodiment of the present invention, the pharmaceutical composition or pharmaceutical kit can reduce the spleen coefficient and inhibit spleen swelling in the subjects with pulmonary fibrosis.

According to the embodiment of the present invention, the pharmaceutical composition or pharmaceutical kit can alleviate the increase of lung coefficient and improve the condition of lung inflammation and fibrosis.

According to the embodiments of the present invention, the drug for treating or alleviating pulmonary fibrosis or improving the symptoms of pulmonary fibrosis can improve the pulmonary ventilation function and/or acid-base balance of the subject with pulmonary fibrosis. Specifically, the combination of oxynidone and dextromethorphan can improve blood pH, partial pressure of oxygen, interstitial fluid residual base, actual bicarbonate, total carbon dioxide, oxygen saturation and/or lactate levels in subjects with pulmonary fibrosis.

According to an embodiment of the present invention, the pharmaceutical composition or pharmaceutical kit can reduce the absolute value of white blood cells, the absolute value of lymphocytes and/or the absolute value of mononuclear cells in the alveolar lavage fluid of a subject with pulmonary fibrosis, to achieve lung anti-inflammatory The role of inflammation.

According to an embodiment of the present invention, the pharmaceutical composition or pharmaceutical kit can reduce NF-KB, TNF-α, IL-2, IL-6, IL-1p and/or or TGF-B levels.

According to an embodiment of the present invention, the pharmaceutical composition or pharmaceutical kit is capable of increasing the IFN-γ level in alveolar lavage fluid of a subject with pulmonary fibrosis.

According to the embodiment of the present invention, the pharmaceutical composition or pharmaceutical kit can improve the inhibitory effect of glucocorticoids on spleen immune cells, and restore the level of spleen immune cells to a normal level.

For example, the spleen immune cells are CD8+T cells, CD4+T cells, B cells.

According to the embodiment of the present invention, the pharmaceutical composition or the pharmaceutical kit can control the pathology of lung tissue structure, for example, reduce the content of hydroxyproline in the subjects with pulmonary fibrosis.

According to an embodiment of the present invention, at the initial stage of pulmonary fibrosis and/or the forming stage of pulmonary fibrosis, the pharmaceutical composition or pharmaceutical kit comprising oxynidone and dextromethorphan can reduce changes in lung tissue structure and inflammatory cell infiltration , Improve pulmonary fibrosis.

According to an embodiment of the present invention, the pharmaceutical composition or pharmaceutical kit is capable of resisting ECM deposition.

For example, the pharmaceutical composition or pharmaceutical kit can reduce bronchial submucosal and pulmonary interstitial collagen fiber deposition.

The present invention also provides a method for treating or alleviating pulmonary fibrosis or improving fibrosis symptoms, comprising administering the above-mentioned pharmaceutical composition or pharmaceutical kit to an individual in need thereof.

The present invention also provides the above pharmaceutical composition or pharmaceutical kit, which is used as a medicine for treating or alleviating pulmonary fibrosis or improving fibrosis symptoms.

The present invention also provides the above-mentioned pharmaceutical composition or pharmaceutical kit, which is used in the method of treating or alleviating pulmonary fibrosis or improving the symptoms of fibrosis.

In the present invention, the pulmonary fibrosis may be idiopathic pulmonary fibrosis (IPF).

According to the present invention, the pharmaceutically acceptable salts include acid addition salts or base addition salts of the compounds of the present invention, such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; sulfuric acid Dialkyl esters such as dimethyl, diethyl, dibutyl, and dipentyl sulfates; long-chain halides such as decyl, lauryl, myristyl, and stearyl chlorides, bromides And iodide; aralkyl halides such as benzyl and phenethyl bromide, etc. By way of example, pharmaceutically acceptable salts include, but are not limited to, hydrochlorides, sulfates, nitrates, bisulfates, hydrobromides, acetates, oxalates, citrates, methanesulfonates, formate or meglumine salts, or alkali metal salts such as sodium, potassium or magnesium salts.

The pharmaceutically acceptable salt of oxynidone is preferably sodium salt.

The pharmaceutically acceptable salt of dextromethorphan is preferably dextromethorphan hydrobromide.

Beneficial effects of the present invention:

Experiments show that the combination of oxynidone or a pharmaceutically acceptable salt thereof and dextromethorphan or a pharmaceutically acceptable salt thereof has excellent effects in repairing lung function, inhibiting inflammation of pulmonary fibrosis and improving pulmonary fibrosis. Specifically, in the mouse model of idiopathic pulmonary fibrosis (IPF) induced by bleomycin (BLM) instillation into the trachea, lung dampness in the combined administration group of oxynidone (F351) and dextromethorphan (DM) Weight and lung coefficient decreased significantly, hydroxyproline content decreased, and pulmonary fibrosis score decreased. Therefore, the pharmaceutical composition or pharmaceutical kit comprising dextromethorphan and oxynidone of the present invention can be used as a medicine for treating or alleviating pulmonary fibrosis or improving symptoms of pulmonary fibrosis.

Description of drawings

Figure 1 shows the body weight change curve of the animals during the experiment.

Figure 2 shows the results of animal survival during the experiment.

Figure 3 shows the results of body weight, lung wet weight and lung coefficient.

Figure 4 shows the results for right lung hydroxyproline content.

Figure 5 shows the HE results of the lung tissues of the mice in each group after 24 hours of modeling (the scales in the figure are all magnified by 200 times).

Figure 6 shows the SR results of the lung tissues of the mice in each group after 24 hours of modeling (the scales in the figure are all magnified by 200 times).

Detailed ways

The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following examples are only for illustrating and explaining the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies realized based on the above contents of the present invention are covered within the scope of protection intended by the present invention.

Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

Example 1

1. Experimental materials

1.1 Test substance

1.2 Solvent

1.3 Molding agent

1.4 Reagents

Sodium carboxymethylcellulose: Sigma, Lot No: SLBJ9393V

Sodium pentobarbital: China Pharmaceutical Group Shanghai Chemical Reagent Company, Lot No: F20021216

PBS: Thermo Fisher Biochemicals (Beijing) Co., Ltd., Hyclone, SH30028.01.B

95% ethanol: Shanghai Reagent Company, article number: 80186961

10% formalin neutral fixative: Xilong Science Co., Ltd., Lot No: 1901232

Hydroxyproline kit: Nanjing Jiancheng Institute of Bioengineering, article number: A030-2-1, production batch number: 20210317, expiration date: 20210916.

1.5 Instruments and equipment

Ordinary optical microscope: Olympus Model: BX53

2. Animals and feeding conditions

2.1 Animals

2.2 Animal reception, health assessment and adaptation period

After the animals arrive at the facility, animal house personnel transfer the animals from their shipping packaging to their cages and inspect each animal. The scope of inspection includes appearance, limbs and cavities, etc., and whether there are any abnormalities in animal posture or movement.

2.3 Environment

Experimental mice were placed in mouse cages (260mm╳160mm╳120mm) in the animal room. The room numbers in which the animals were housed were recorded in the experimental records throughout the experiment. Mice were littered with high-temperature-treated corn cobs, and the litter was changed twice a week. The room in which the experimental mice were housed was located in a quasi-SPF animal room with filtered ventilation in this area at a frequency of 15-25 air changes per hour. The temperature is maintained between 20-25°C (68-77°F) and the relative humidity is 40-70%. The lighting conditions were 12 hours (08:00-20:00) daylight irradiation and 12 hours darkness per day.

2.4 Food and Drinking Water

Experimental mice had unlimited access to rodent-specific feed (which had been irradiated and sterilized). Each batch of animal feed received is accompanied by a certificate of analysis for the corresponding batch from the supplier.

During the whole experiment, the experimental mice had unlimited access to high-temperature sterilized municipal tap water. The adaptation period before the start of the experiment was 30 days.

The levels of known contaminants in animal food and drinking water within the foreseeable range will not interfere with the purpose or conduct of this experiment.

2.5 Cage and animal identification

Each animal is assigned a unique number. Animals were numbered and identified by the method of cutting toes. The numbering rules were as follows: starting from the rear left foot, the number 1 was cut to the left, the number 2 was cut to the left 2, the number 3 was cut to the left 3, and the fifth toe was cut in order to be number 5. Before the animals are grouped, the animal species/strain, gender, cage number, experimental items, drug administration, experiment start date and other information are marked on the label of the mouse cage.

After the animals are grouped, the mouse cages are labeled with the group information and the above information. The grouping situation is recorded in the random grouping file. Cages were used to layer the mouse cages to reduce the impact of environmental factors on the experiment.

3. Experimental methods and procedures

3.1 Animal grouping

The mice were randomly divided into 5 groups according to body weight, and the grouping and treatment conditions are shown in Table 1.

Table 1 Animal Grouping Treatment Scheme

Remarks: DM (sc, tid), F351 (P.O, qd), it: intratracheal injection.

3.2 Modeling

Mice were anesthetized with sodium pentobarbital at a dose of 65 mg/kg. With the assistance of LED cold light, 76 μL of BLM (concentration corresponding to the group) solution or corresponding volume of normal saline was slowly injected through the trachea. Immediately turn the mouse upright and turn it left and right, so that the drug solution is evenly distributed in the lungs.

3.3 Administration

F351 and 0.5% cmc were administered by intragastric administration once a day at around 8:30 am; DM was administered subcutaneously three times a day at 8:30 am, 12:00 noon and 16:00 pm respectively.

3.4 Tissue collection

24 hours after the end of administration, the body weight of the surviving animals was weighed. Animals were anesthetized by intraperitoneal injection of pentobarbital sodium and sacrificed by exsanguination from the abdominal aorta. The lungs were clipped and weighed wet. The left lung of the mouse was directly fixed in 10% neutral formalin solution for more than 24 hours before subsequent treatment; the right lung was immediately frozen at -80°C for detection of hydroxyproline.

3.5 Observation indicators

3.5.1 Animal general condition, body weight and mortality

From the first day of administration, the appearance, behavioral activities and fecal properties of each mouse were observed every day. All abnormal appearance and behavior were recorded on the clinical observation form. Weigh your body weight twice a week. If your body weight changes drastically, you may need to measure your body weight if necessary.

3.5.2 Lung coefficient

Lung coefficient is an index that reflects the proportional relationship between lung mass and body mass. Wet lung weight and body weight are weighed by electronic balance respectively. The calculation method of lung coefficient is (wet lung weight mg/body weight g).

3.5.3 Determination of hydroxyproline content

The content of collagen in tissue can be reflected by the content of hydroxyproline. The right lung wet tissue of the mouse was weighed, and the content of hydroxyproline in the lung tissue was determined according to the kit instructions.

1. Measuring principle:

The oxidation product produced by hydroxyproline under the action of oxidizing agent and dimethylaminobenzaldehyde is purple-red, and its content can be deduced according to the depth of its color.

2. Kit composition and preparation: (50T/48 samples)

Reagent 1: powder x 1 bottle, solution A 10ml x 1 bottle, solution B 20ml x 1 bottle, before use, add 10ml of solution A to the powder and fully dissolve it (seeing from the inside of the bottle mouth, it is completely dissolved), and then add B 20ml solution and mix well. The prepared reagents are valid for 3 months when stored in a refrigerator at 4°C to 8°C.

Reagent 2: Liquid 30ml x 1 bottle, stored in a refrigerator at 4°C in the dark for 3 months.

Reagent 3: powder × 1 bottle, solvent 30ml × 1 bottle, before use, add one powder to 30ml solvent to fully dissolve, after preparation, store in a refrigerator at 4°C in the dark for 1 month.

Reagent 4: Standard product 5mg×3 vials, stored in 4°C refrigerator for 6 months.

Preparation of 100μg/ml standard stock solution: before testing, dissolve a standard product in double distilled water, dilute it to 50ml, and store it in a refrigerator at 4°C for 2 weeks.

Preparation of 5μg/ml standard application solution: Take 1ml of 100μg/ml standard stock solution and add double distilled water to make up to 20ml.

3. Determination of hydroxyproline:

(1) Sample pretreatment reagent (stored at 4°C): (50T)

1. Hydrolyzate 60ml x 1 bottle: 2. Indicator 5ml x 1 bottle: 3. Adjust pH A solution 60ml x 1 bottle;

4. pH adjustment solution B 30ml x 1 bottle: 5. Activated carbon x 1 bag.

(2) Sample pretreatment:

1. Sample hydrolysis:

① Serum (syrup): Take 0.5ml of serum (serum) and accurately add 1ml of hydrolyzate, and mix well. Put it in the test tube and cover it, and hydrolyze it for 20 minutes at 95°C or in a boiling water bath.

②Urine (culture solution): Take 1.0ml of urine (culture solution) and add 1ml of hydrolyzate, mix well. Put it in the test tube and cover it, and hydrolyze it for 20 minutes at 95°C or in a boiling water bath.

③Tissue: Accurately weigh 30-100 mg of tissue wet weight and put it into a test tube, accurately add 1 ml of hydrolyzate, and mix well. After capping, hydrolyze at 95°C or in a boiling water bath for 20 minutes (mix once after 10 minutes of hydrolysis to make the hydrolysis more complete).

2. Adjust the pH value to about 6.0-6.8.

① After cooling each test tube with running water, add 10 μl of indicator to each tube and shake well;

② Accurately add 1.0ml of pH adjusting solution A to each tube and mix well (the solution should be red at this time);

③Use a 200μl sampler to draw the pH-adjusting solution B into each tube, carefully add the pH-adjusting solution drop by drop, and mix well after each drop* until the color of the indicator in the liquid turns yellow-green** ( That is, when the red color disappears).

At this time, the pH value is around 6.0-6.8 (approximately add about 100μl to 500μl of pH adjusting solution B); Ground test tubes can be replaced by ordinary glass test tubes, and plastic film or refrigerator plastic wrap can be used to press the test tube mouth when mixing each time, and fully vortex to mix.)

④ Then add double distilled water to 10ml and mix well;

⑤ Take 3-4ml of the diluted hydrolyzate and add an appropriate amount of activated carbon (about 20-30mg, the supernatant is clear and colorless after centrifugation), mix well, centrifuge at 3500 rpm for 10 minutes, and carefully take 1ml of the supernatant for testing.

(3) Operation table

the	blank tube	standard tube	Assay tube
double distilled water	1.0	the	the
5μg/ml standard application solution	the	1.0	the
Detection solution (ml)	the	the	1.0
Reagent 1 (ml)	0.5	0.5	0.5

Mix well and let stand for 10 minutes

Reagent 2 (ml)

0.5

0.5

0.5

Mix well and let stand for 10 minutes

Reagent 3 (ml)

0.5

0.5

0.5

Mix well, put in a water bath at 60°C for 15 minutes, after cooling, centrifuge at 3500 rpm for 10 minutes, take the supernatant at a wavelength of 550nm, 1cm optical path, adjust to zero with double distilled water, and measure the absorbance of each tube.

4. Calculation formula

(1) Serum (serum) calculation formula:

1. Calculation formula:

(2) The calculation formula of urine:

1. Calculation formula:

(3) The calculation formula of the organization:

1. Calculation formula:

3.5.4 Lung histopathological examination (HE staining, light microscope analysis of lung structure).

H&E staining: Paraffin sections were routinely dewaxed to water, dipped in hematoxylin staining solution for 10 minutes, rinsed in tap water, differentiated with 1% hydrochloric acid alcohol for 10 seconds, rinsed in tap water, turned blue with 2% sodium bicarbonate solution for 10 seconds, rinsed in tap water, dipped in 1% eosin solution Rinse with tap water for 2 minutes, dehydrate with 95% alcohol, make transparent in xylene, and observe under a light microscope after mounting with neutral resin.

4. Statistical analysis

Chi-square test was used for the mortality rate; the results of measurement data were expressed as mean ± standard deviation, and Dunnett’s multi-comparison method was used for pairwise comparison between groups; Mann-Whitney non-parametric test was used for grade data between groups. A p<0.05 will be considered a statistically significant difference.

5. Experimental results

5.1 Animal general condition, body weight and mortality

At the beginning of the experiment, the animals in each group had bright fur, normal activities and good condition. As shown in Figure 1, the mice in the control group had normal body weight, good activity and state throughout the experiment. Compared with the control group, the mice in the model group (BLM/0.5% cmc (n=15)) and each administration group showed weight loss. Compared with the model group, the body weight of the mice in the DM+F351 group (BLM/DM-10npk+F351-100mpk (n=15)) tended to decrease, and there was no significant difference in the weight changes of the mice in the other administration groups compared with the model group.

As shown in Figure 2, except that the control group and the DM-10ng/kg group (dextromethorphan group) did not die, the animals in other administration groups all died, and there was no significant difference compared with the model group.

5.2 Lung wet weight

As shown in Figure 3, compared with the control group, the lung wet weight and lung coefficient increased significantly in the model group. Compared with the model group, the lung wet weight and lung coefficient decreased significantly in the DM+F351 group, and there was no significant difference in the other administration groups.

5.3 Hydroxyproline content

As shown in Figure 4, compared with the control group, the content of hydroxyproline in the model group increased significantly. Compared with the model group, each administration group had a downward trend, and compared with the DM-10ng/kg group (dextromethorphan group) and the F351-100mg/kg (oxynidone group) used alone, the DM+F351 group (oxynidone dextromethorphan combined group) has a downward trend.

5.4 Histopathological examination

Figure 5 shows the HE results of the lung tissues of mice in each group after 24 hours of modeling. As can be seen from the figure,

The lung tissue structure of mice in the control group was clear, there was no obvious inflammatory cell infiltration around the bronchi and blood vessels, and no obvious thickening of the tube wall. The alveolar structure was intact, and the alveolar septum was not significantly thickened. The lung tissue structure of the mice in the model group changed significantly, the lung tissue structure was destroyed, the alveolar septum was thickened, a large number of inflammatory cells infiltrated, part of the alveolar space disappeared, and replaced by mononuclear inflammatory cells and fibrous tissue. sex fibroblasts.

In the DM-10ng/kg group (dextromethorphan group), the lung tissue structure of the mice was destroyed, the alveolar septum was thickened, a large number of inflammatory cells infiltrated, and part of the alveolar cavity disappeared, replaced by mononuclear inflammatory cells and fibrous tissue. sex fibroblasts.

The overall lung structure of the F351-100mg/kg group (oxynidone group) was slightly better than that of the model group, some alveolar septa were thickened, fibrous foci formed, accompanied by inflammatory cell infiltration, alveolar wall structure was destroyed, and some alveoli disappeared.

In the DM+F351 group (oxynidone and dextromethorphan combined group), the alveolar septum was thickened, inflammatory cells infiltrated, fibrous deposits were seen, distributed in a focal shape, the structure of the alveolar wall was destroyed, and some alveoli disappeared.

Figure 6 shows the SR results of the lung tissues of mice in each group after 24 hours of modeling. As can be seen from the figure,

The lung tissue structure of the mice in the control group was clear, the alveolar septa were slightly thickened in a small area, and there were slightly more red collagen fibers. The lung tissue structure of the mice in the model group changed significantly, the alveolar structure was destroyed, fibrous foci appeared, and there were more SR-stained red collagen fibers in the alveolar septum.

In the DM-10ng/kg group (dextromethorphan group), the alveolar septum was thickened, the structure was destroyed, fibrous foci appeared, and there were more red collagen fibers.

The alveolar structure of mice in F351-100mg/kg group (oxynidone group) was complete, and there were more collagen fibers stained red by SR in the alveolar septum.

In the DM+F351 group (oxynidone and dextromethorphan combined group) the alveolar septum was thickened, the structure was destroyed, fibrous foci appeared, and a small amount of red collagen fibers existed.

Pulmonary fibrosis score: Pulmonary fibrosis score (0-8) is evaluated with reference to the changes in the degree of fibrosis in the lung septum, alveoli and the whole. The specific results are shown in Table 1.

Table 1 Pulmonary fibrosis score

Histopathological examination found that compared with the control group, the degree of lung lesions in the model group was significantly aggravated. Compared with the model group, the fibrosis scores of the mice in the DM+F647 group showed a downward trend, and there was no significant difference in the fibrosis scores of the mice in the other administration groups. The specific results are shown in Table 2.

Table 2 Grades of pulmonary fibrosis

6. Experimental conclusion

(1) Compared with the control group, the body weight of the animals in the model group and each administration group decreased significantly.

(2) Compared with the control group, the death rate in the model group increased significantly. Except that no animal died in the DM-10ng/kg group, the animals in the other administration groups all died, and there was no significant difference compared with the model group.

(3) Compared with the control group, the wet lung weight and lung coefficient of the model group increased significantly, while the wet lung weight and lung coefficient of the DM+F351 group decreased significantly.

(4) Compared with the control group, the hydroxyproline content in the right lung of the model group and each treatment group was significantly increased. Compared with DM-10ng/kg group and F351-100mg/kg alone, the right lung hydroxyproline content of DM+F351 group tended to decrease.

(5) Compared with the control group, the pulmonary fibrosis score of the model group increased significantly. Compared with the model group, the pulmonary fibrosis score in the DM+F351 group showed a downward trend.

Example 2

Compared with Example 1, the difference is that the administration method of the combined administration group of oxynidone (F351) and dextromethorphan (DM) is: both F351 and DM are given by intragastric administration, once a day, at 8 am. : about 30; dosage: DM-10mpk+F351-100mpk.

Oxynidone (F351) and dextromethorphan (DM) combined administration group in this embodiment have the same therapeutic effect on mice as in Example 1 and oxynidone (F351) and dextromethorphan (DM) combined administration group quite.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above-mentioned embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. A pharmaceutical composition, characterized in that it comprises oxynidone represented by formula (I) or a pharmaceutically acceptable salt thereof, and dextromethorphan or a pharmaceutically acceptable salt thereof,

   
2. A pharmaceutical kit, characterized in that it comprises oxynidone represented by formula I or a pharmaceutically acceptable salt thereof and dextromethorphan or a pharmaceutically acceptable salt thereof, which are placed separately.
3. The pharmaceutical composition as claimed in claim 1 or the pharmaceutical kit as claimed in claim 2, wherein the combination of oxynidone or a pharmaceutically acceptable salt thereof and dextromethorphan or a pharmaceutically acceptable salt thereof The mass ratio can be (1-10000):(1-5000), for example (10-8000):(1-3000), (10-6000):(1-2000), (10-5000):(1 -1000), (50-4000): (10-500), (100-2000): (50-300), (100-1000): (10-100), (200-1000): (100-200 ), (300-800):(100-200), (300-500):(120-180), (1000-5000):(1-800), (2000-5000):(1-600), (2000-5000): (1-500), (2000-5000): (1-100), (2000-5000): (1-50), (3000-5000): (1-30), (10 -5000):(100-5000),(20-4000):(500-4000),(30-3000):(1000-3000),(50-2000):(1000-3000),(60-1000 ):(1000-3000) or (80-800):(1000-3000).
4. The pharmaceutical composition or pharmaceutical kit according to any one of claims 1-3, further comprising at least one pharmaceutically acceptable auxiliary material.

   Preferably, the pharmaceutically acceptable excipients are various pharmaceutical excipients known in the field of pharmacy, including but not limited to: diluents, binders, antioxidants, pH regulators, preservatives, lubricants, disintegrators agent etc.
5. The pharmaceutical composition or pharmaceutical kit according to any one of claims 1-4, wherein the diluent is selected from lactose, starch, cellulose derivatives, inorganic calcium salts, sorbitol and the like.

   Preferably, the binder is selected from starch, gelatin, sodium carboxymethylcellulose, polyvinylpyrrolidone and the like.

   Preferably, the antioxidant is selected from vitamin E, sodium bisulfite, sodium sulfite, butylated hydroxyanisole and the like.

   Preferably, the pH regulator is selected from hydrochloric acid, sodium hydroxide, citric acid, tartaric acid, Tris, acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate and the like.

   Preferably, the preservative is selected from methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, m-cresol, benzalkonium chloride and the like.

   Preferably, the lubricant is selected from magnesium stearate, micronized silica gel, talcum powder and the like.

   Preferably, the disintegrant is selected from starch, methylcellulose, xanthan gum, cross-linked sodium carboxymethylcellulose and the like.
6. The pharmaceutical composition or pharmaceutical kit according to any one of claims 1-5, wherein the dosage form of the pharmaceutical composition or pharmaceutical kit can be an oral agent, such as tablet, capsule, pill, powder, granule formulations, suspensions, syrups, etc.; parenteral administration (eg, injection) formulations, such as injections, powder injections, etc., which can be administered intravenously, intraperitoneally, subcutaneously or intramuscularly.
7. Use of the pharmaceutical composition or pharmaceutical kit according to any one of claims 1-6 in the preparation of medicines for treating or alleviating pulmonary fibrosis or improving symptoms of pulmonary fibrosis.
8. The application according to claim 7, characterized in that the pharmaceutical composition or pharmaceutical kit can restore lung function, inhibit inflammation of pulmonary fibrosis and/or improve fibrosis.

   Preferably, the pharmaceutical composition or pharmaceutical kit can reduce the spleen coefficient and inhibit spleen swelling in subjects with pulmonary fibrosis.

   Preferably, the pharmaceutical composition or pharmaceutical kit can alleviate the increase of lung coefficient and improve the condition of lung inflammation and fibrosis.

   Preferably, the drug for treating or alleviating pulmonary fibrosis or improving the symptoms of pulmonary fibrosis can improve the pulmonary ventilation function and/or acid-base balance of subjects with pulmonary fibrosis.

   Preferably, the pharmaceutical composition or pharmaceutical kit can reduce the absolute value of leukocytes, absolute values of lymphocytes and/or absolute values of monocytes in alveolar lavage fluid of subjects with pulmonary fibrosis, so as to achieve the effect of anti-inflammation in the lungs.

   Preferably, the pharmaceutical composition or pharmaceutical kit can reduce NF-KB, TNF-α, IL-2, IL-6, IL-1p and/or TGF-B in alveolar lavage fluid of subjects with pulmonary fibrosis Level.

   Preferably, the pharmaceutical composition or pharmaceutical kit can increase the IFN-γ level in alveolar lavage fluid of subjects with pulmonary fibrosis.

   Preferably, the pharmaceutical composition or pharmaceutical kit can improve the inhibitory effect of glucocorticoids on spleen immune cells, and restore the level of spleen immune cells to a normal level.

   Preferably, the pharmaceutical composition or pharmaceutical kit is capable of controlling lung tissue structural lesions, such as reducing the content of hydroxyproline in subjects with pulmonary fibrosis.

   Preferably, at the initial stage of pulmonary fibrosis and/or the forming stage of pulmonary fibrosis, the pharmaceutical composition or pharmaceutical kit comprising oxynidone and dextromethorphan can reduce changes in lung tissue structure and infiltration of inflammatory cells, and improve lung fibrosis. status.

   Preferably, the pharmaceutical composition or pharmaceutical kit is resistant to ECM deposition.

   Preferably, the pharmaceutical composition or pharmaceutical kit can reduce bronchial submucosal and pulmonary interstitial collagen fiber deposition.
9. A method for treating or alleviating pulmonary fibrosis or improving symptoms of fibrosis, comprising administering the pharmaceutical composition or pharmaceutical kit according to any one of claims 1-6 to individuals in need thereof.
10. The pharmaceutical composition or pharmaceutical kit according to any one of claims 1-6, which is used as a medicine for treating or alleviating pulmonary fibrosis or improving fibrosis symptoms.

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